De Novo Accumulation of Tetrodotoxin and Its Analogs in Pufferfish and Newt and Dosage-Driven Accumulation of Toxins in Newt: Tissue Distribution and Anatomical Localization

Abstract

The present study was undertaken to determine the amounts of tetrodotoxin (TTX) and its analogs (TTXs) in various tissues of toxin-bearing pufferfish (Canthigaster revulata and Takifugu flavipterus) and newt (Cynops pyrrhogaster) using specific polyclonal antibodies against TTXs, and to compare the obtained results with those mainly determined by high-performance liquid chromatography with fluorescence detection (HPLC-FLD). The anatomical localization of TTXs in these animals was also demonstrated immunohistochemically using the above-mentioned antibody. The ratio of the total amount of TTXs determined by ELISA to that determined by HPLC-FLD changed depending on the tissues examined in pufferfish. Such differences were also observed with the newt in tissue- and individual-dependent manners. Furthermore, TTXs, as well as decarbamoylsaxitoxin (dcSTX), an analog of saxitoxin (STX), were traced for their dynamic changes in tissue distribution, when the newt was fed authentic toxins or toxic animal tissues exogenously, demonstrating that a TTX analog, 5,6,11-trideoxyTTX, and dcSTX were not metabolized into TTX or STX. TTXs-immunoreactive (ir) staining was observed in the pancreas region of the hepatopancreas, the oocytes at the perinucleolus stage, the sac-like tissues just outside the serous membrane of the intestine, and the gland-like structure of the skin, but not in the muscles of pufferfish. TTXs-ir staining was also detected in the mature glands in the dermis of the adult and regenerated tail, but not in the liver, intestine, testis and ovary of the adult newt. TTXs-ir staining was detected in the epithelial cells of the intestine, the ovary, the mucous cells, and the dermis of the TTXs-administered newt. These results suggest that TTXs absorbed from the environment are distributed to various organs or tissues in a species-specific manner, regardless of whether or not these are metabolized in the bodies of toxin-bearing animals.